Project Summary ? Project 3 There is an urgent need to develop and validate novel pre-clinical models of human diarrheal diseases. Recently, human intestinal enteroid (HIE) cultures have been created to accelerate in vitro investigations of intestinal pathophysiology and to discover new therapies. These human adult stem cell derived cultures are more physiologically faithful than monolayer cell culture or animal models, but their application to study a large variety of intestinal commensals and enteropathogens is limited. The apical sides of epthelial cells in HIEs are not easy for pathogens to access since they are located in the interior. In addition, current HIE culture systems lack the ability to apply mechanical stimulation, which is physiologically relevant. Further, the main scaffold material used to grow enteroids is Matrigel, which is softer mechanically than actual biological tissues. Project 3 involves a collaborative goal to develop tailored biomaterial platforms for HIEs that can be mechanically stimulated and that will promote cell and tissue polarity as well as differentiation through the villus-crypt axis. The development of new HIE platforms will facilitate enteropathogen infection of cells, and allow physiologic study of interactions of endothelium with the microbiome, inflammatory cells, or the sub- endothelial intestinal tissue. These will also provide new models for future testing of drugs that can access the apical side of the epithelium. These engineered platforms will be opportunities for synergistic interactions between projects and cores of the overall proposal; screen-based analyses of the biology of commensal- stimulated or viral/bacterial pathogen-stimulated intestinal epithelium will reveal information about critical cell- matrix interactions that can be incorporated into the biomaterial platform, and as the various biomaterial platforms are developed, they can be implemented within Projects 1-2. These platforms are also planned to integrate with ubiquitous equipment to facilitate broad distribution to infectious disease labs. Our collaborative team brings together expertise with native and synthetic biomaterials as scaffolds to direct cell behavior, developing experimental frameworks for mechanically stimulating cells and tissues, promoting vasculogenesis in engineered tissues, and culturing HIEs. We propose the following specific aims: 1. To examine the roles of independent, physiologically relevant mechanical cues on HIE culture, epithelial cell differentiation, and pathogenic infection within HIEs, we will culture HIE fragments atop and within collagen/Matrigel hydrogels and employ mechanical stimulation bioreactors to impose cyclic stretch and shear. 2. To develop a tailored, anatomically faithful model for the intestinal epithelium, we will generate synthetic biomaterial platforms that drive the formation of a polarized epithelium differentiated through the villus-crypt axis, and systematically investigate the roles of adhesive ligands, topography, and substrate stiffness. 3. To determine how other villus cells promote epithelial cell differentiation and function within intestinal culture models, we will encapsulate mesenchymal and microvascular cells within the biomaterial platform.